This proposal is to determine the gene regulatory mechanisms that guide hematopoietic progenitor cells through the process of T-lineage commitment in the thymus. It focuses on the roles of two powerful regulators, the Notch/Delta signaling system and the transcription factor PU.1. Both of these factors are essential for the initiation of T-cell development from multipotent hematopoietic precursors, but their roles become divergent as the cells approach full T-lineage commitment. Notch/Delta signaling not only initiates T-cell specification, but also continues to support viability and expression of T-lineage specific genes throughout the commitment process. PU.1 expression and activity appear to maintain developmental plasticity in the early precursors, so that the cells do not become fully committed to the T lineage until PU.1 expression is finally shut off. The first aim of this revised competitive renewal proposal is based on new data showing that Notch/Delta signaling dramatically modulates the spectrum of PU.1 transcriptional activities in immature thymocytes so as to block lineage diversion selectively. The second aim is based on new insights into mechanisms that may close the door on alternative fates by silencing PU.1. In our first specific aim, we plan to determine exactly how Notch/Delta signaling is harnessed in pro-T cells to constrain PU.1 effects on lineage choice: through effects on the PU.1 protein itself, or through effects on the competing genetic cascades that execute lympho-myeloid lineage decisions. The second aim is based on our new data that locate a potential T lineage-specific silencing element in a previously uncharacterized cis-regulatory region of the PU.1 gene. We propose to map the silencing site(s) precisely and use it to identify the rate-limiting repressor proteins that engage it to shut PU.1 off during T lineage commitment. Through dissection of the role of this element in vivo and vitro and the factors that act on it, a novel regulatory component of the T-lineage commitment mechanism should be revealed. The process of T lineage commitment is intimately linked with the correct operation of tumor suppressive functions in the thymus. Both Notch and PU.1 as well as the genes they affect must be correctly regulated during commitment in order to avoid leukemic transformation. The mechanisms studied here imply that mixed-lineage thymic lymphomas may also result from primary defects of a natural T/myeloid lineage choice.